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The Journal of Immunology, 1998, 161: 1751-1757.
Copyright © 1998 by The American Association of Immunologists

ß2-Microglobulin-Dependent T Cells Are Not Necessary for Alloantigen-Induced Th2 Responses After Neonatal Induction of Lymphoid Chimerism in Mice1

Gilles Foucras, Christiane Coureau, Leo Beijleveld, Philippe Druet, Abdelhadi Saoudi and Jean-Charles Guéry2

Institut National de la Santé et de la Recherche Médicale (INSERM) U.28, Université Paul Sabatier, Hôpital Purpan, Toulouse Cedex, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We have analyzed the requirement for ß2-microglobulin (ß2m)-dependent T cells in the generation of allogeneic Th2 responses in vivo. A neonatal injection of semiallogeneic cells in BALB/c mice induces a state of chimerism that promotes the differentiation of donor-specific CD4+ T cells toward the Th2 phenotype. Polyclonal T-B cell interactions occur in this model between host Th2 and donor B cells, resulting in the production of IgE Abs. IgE production and Th2-priming are critically dependent upon the early production of IL-4. Our data in the present paper demonstrate that: 1) IgE synthesis and the up-regulation of MHC class II and CD23 molecules on B cells are independent of ß2m expression in the host, 2) no difference in the induction of CD4 alloreactive Th2 cells could be observed between ß2m-/- and their wild-type control littermates when Th2-priming was measured in adult mice, and 3) the Th2 response and IgE production is induced in the complete absence of ß2m-dependent T cells both in the host and in the inoculum. Therefore, using a variety of assays, we could not demonstrate diminished responses in mice with a disrupted ß2m gene in this model of Th2-mediated allogeneic interaction, indicating that ß2m-dependent NK1.1+ and CD8+ T cells are not required for the generation of alloreactive Th2 responses in vivo.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The CD4+ T cells have been subdivided into distinct subsets according to their cytokine profile (1, 2, 3). Mouse Th1 cells mainly produce IFN-{gamma}, TNF-ß, and IL-2, and are responsible for cell-mediated immunity, whereas Th2 cells secrete IL-4, IL-5, IL-10, and IL-13, which provide help to B cells resulting in IgG1 and IgE production. T cells that produce a mixture of both Th1- and Th2-associated cytokines have been called Th0 (4). Studies in TCR-transgenic mice have shown that Th1 or Th2 effector cells can arise from the same precursor CD4+ T cells, and that this process is mainly influenced by the cytokine environment during the initial phase of T cell activation (5, 6). Indeed, IL-12 and IL-4 have been shown to play a decisive role by driving the polarization of T cell responses toward the Th1 or Th2 phenotype, respectively (5, 6, 7).

It is now well-established that IL-4 is the principal physiologic regulator of the differentiation of naive CD4+ T cells toward IL-4-producing Th2 cells in vivo (8, 9, 10). Thus, determining the cellular origin of the initial burst of IL-4 as well as how this production is regulated is central to understanding the genesis of Th2 responses. Among the potential candidates, the cells that have drawn the most attention are NK1.1+ T cells, which are a rare T cell population that is restricted by ß2-microglobulin (ß2m)3-associated, nonclassical MHC molecules (11). Due to their capacity to produce IL-4 upon an injection of anti-CD3 in vivo, NK1.1+ T cells have been proposed as the IL-4-providing cells at the onset of Th2 responses (12). Nevertheless, the physiologic importance of this T cell subset has been seriously challenged by several reports demonstrating that Th2 responses to a variety of infectious agents or protein Ags were normal in ß2m-deficient mice that lack the NK1.1+ T cell population (13, 14, 15).

So far, the role of NK1.1+ T cells has been ascertained in only one model of Th2 response, which is the induction of IgE production induced by the administration of mouse IgD-specific goat Abs (12). The administration of this polyclonal stimulant induces a strong IgE response in various strains; this response is associated with the priming of Th2 cells that are specific for goat IgG proteins (12, 16). In this model, it has been suggested that Ag presentation by activated B cells stimulates T cells to produce IL-4 (16). Indeed, in vivo and in vitro studies have shown that targeting Ag presentation to B cells favors Th2 cell development (17, 18, 19). Since direct evidence for the involvement of NK1.1+ T cells in the induction of an IL-4-dependent response has only been reported in the anti-IgD model, we were interested in determining whether a similar mechanism would be at play in other models of polyclonal T-B cell interactions, such as Th2 responses in allogeneic reactions (20). An injection of semiallogeneic spleen cells (SCs) in newborn BALB/c mice activates host-derived alloreactive Th2 cells that react with MHC class II alloantigens on donor B cells. This cognate interaction induces a polyclonal B cell activation that is characterized, like the anti-IgD system, by increased levels of IgG1 and IgE (21, 22).

To evaluate the role of NK1.1+ T cells in this model, we first analyzed the alloreactive Th2 response and IL-4-dependent IgE production in ß2m-deficient BALB/c mice that had been injected at birth with semiallogeneic (BALB/c x C57BL/6)F1 (CB6 F1) SCs. Although the presence of donor T cells in the inoculum is not necessary for the establishment of neonatal tolerance and for the activation of CB6 F1 B cells (23), we also tested the Th2 response that was induced in ß2m-/- mice by the injection of splenocytes from ß2m-deficient CB6 F1 mice. Our data demonstrate that IL-4-dependent IgE production can be induced in the complete absence of ß2m-dependent T cells, indicating that NK1.1+ T cells and CD8+ T cells are dispensable for alloreactive Th2 cell responses.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice, neonatal injection of semiallogeneic SCs, and immunization

BALB/c (H-2d), C57BL/6 (H-2b) and CB6 F1 mice were purchased from Centre d’Elevage R. Janvier (Le Genest St. Isle, France). H-2b mice with disrupted ß2m genes (24) were back-crossed to BALB/c mice as described previously (13). ß2m-/- mice on a BALB/c or C57BL/6 background were used after nine back-crosses. H-2d ß2m-/- mice at the seventh back-cross were a kind gift of Dr. Luciano Adorini (Roche Milano Ricerche, Milano, Italy). ß2m-/- mice in the C57BL/6 background were initially obtained from the Centre National de la Recherche Scientifique (Centre de Développement des Techniques Avancées, Orléans, France). BALB/c ß2m-/-, C57BL/6 ß2m-/- and CB6 F1 ß2m-/- mice were bred and maintained in our own animal facility.

BALB/c ß2m+/- and BALB/c-/- littermates were injected i.p. with 108 CB6 F1 SCs within the first 24 h of life. Control animals were left untreated. In some experiments, adult mice were immunized s.c. in the hind footpads with 60 x 106 irradiated (2400 rad) SCs from allogeneic C57BL/6 mice. At 5 to 6 days after immunization, the draining popliteal and inguinal lymph nodes were removed and further processed as described below.

Quantitation of serum IgE

The IgE concentration was determined in serum by a sandwich ELISA. Briefly, polyvinyl microtiter plates (Falcon 3012, Becton Dickinson Labware, Oxnard, CA) were coated with 50 µl of mouse IgE-specific LO-ME-3 rat mAb (LO/IMEX, University of Louvain, Brussels, Belgium) in PBS. Bound IgE was revealed using biotinylated R35–92 IgE-specific rat mAb (PharMingen, San Diego, CA) with similar results. The bound biotinylated anti-IgE Abs were revealed by an additional 30-min incubation with alkaline phosphatase-conjugated streptavidin (Jackson Immunoresearch Laboratories, Avondale, PA). The plates were incubated with the developing substrate p-nitrophenylphosphate disodium (Sigma, St. Louis, MO) in diethanolamine buffer (pH 9.6), and absorbance was read at 405 nm with an automated microplate ELISA reader (Emax, Molecular Devices, Sunnyvale, CA). The IgE concentration was quantified from three titration points using a purified mouse IgE mAb, IgE-3 (PharMingen), to generate standard curves.

Flow cytometric analysis

SCs were incubated with optimal concentrations of FITC-, PE-, or biotin-conjugated mAb for 20 min at 4°C in PBS containing 5% FCS and 0.1% sodium azide. The following mAbs were used: FITC-conjugated 14.4.4S anti-I-E (HB 32; American Type Culture Collection (ATCC), Manassas, VA), biotin-6B2 anti-B220 (PharMingen), biotin-B3B4 anti-CD23, and biotin-Y-3 anti-Kb (HB 176, ATCC). Cells were then washed and stained with streptavidin-CyChrome (PharMingen). Data were collected on 10,000 cells as determined by forward and side light scatter intensity on an XL Coulter cytometer (Coultronics, Maegency, France) and analyzed using CellQuest software (Becton Dickinson, Mountain View, CA).

T cell assays

For cytokine production analysis, lymph node cells (LNCs) from mice immunized with allogeneic SCs were cultured at 3 x 105 cells/well in 96-well culture plates (Costar, Cambridge, MA) in the presence of 3 x 105 cells/well of irradiated SCs of the indicated origin. The culture medium used was RPMI 1640 (Life Technologies, Cergy Pontoise, France) supplemented with 5% FCS (ATGC Biotechnologie, Noisy Le Grand, France), 1% pyruvate, 1% nonessential amino acids, 1% L-glutamine, 50 µM 2-ME, and 50 µg/ml gentamycin (Sigma). Cultures were incubated for 3 days in a humidified atmosphere of 5% CO2 in air. Supernatants from replicate cultures (usually 3–4 wells) were collected after 72 h and pooled for cytokine analysis. For T cell proliferation assays, cell cultures were pulsed for 8 h with 1 µCi of [3H]TdR (40 Ci/nmol, the Radiochemical Centre, Amersham, Little Chalfont, U.K.) before harvesting on a glass fiber filter. The incorporation of [3H]TdR was measured by direct counting using an automated beta-plate counter (Matrix 9600, Packard, Meriden, CT).

Cytokine assays

IFN-{gamma}, IL-4, IL-5, and IL-10 were quantified by a two-site sandwich ELISA as described previously (13). For IFN-{gamma}, polyvinyl microtiter plates (Falcon 3012) were coated with 100 µl of anti-IFN-{gamma} AN-18.17.24 mAb (25) in carbonate buffer. After blocking, samples (50 µl/well) that had been diluted in test solution (PBS containing 5% FCS and 1 g/l phenol) were incubated together with 50 µl of peroxidase-conjugated XMG1.2 IFN-{gamma}-specific mAb (2). After an overnight incubation at 4°C, bound peroxidase was detected by 3,3'-5,5'-tetramethylbenzidine (Fluka Chemie AG, Buchs, Switzerland), and adsorbance was read at 450 nm with an automated microplate ELISA reader (Emax, Molecular Devices). For IL-4, IL-5, and IL-10 determination, two-site ELISAs were performed with paired mAbs (all purchased from PharMingen). Cytokines were quantified from two to three titration points using standard curves that had been generated with purified recombinant mouse cytokines, and results were expressed as the cytokine concentration in nanograms or in picograms per milliliter. The detection limits were 15 pg/ml for IFN-{gamma} and IL-4, 3 pg/ml for IL-5, and 30 pg/ml for IL-10.

Flow cytometric analysis of intracellular cytokine synthesis

LNCs were cultured with allogeneic, irradiated SCs from C57BL/6 ß2m-/- mice as indicated above. After 72 h of culture, cells were harvested, washed, and recultured for an additional 72 h in complete medium. After Ficoll separation, living cells were collected, resuspended at 106/ml, and stimulated with PMA (Sigma, 50 ng/ml) plus ionomycin (Sigma, 1 µg/ml) for 4 h. At 2 h before cell harvest, 10 µg/ml of brefeldin A (Sandoz, Basel, Switzerland) was added. Cells were harvested, washed in the presence of brefeldin A, and stained using biotinylated anti-CD4 mAb (PharMingen) followed by streptavidin-CyChrome (PharMingen). Labelled cells were then fixed with 2% paraformaldehyde (Fluka). Intracytoplasmic staining was performed as described previously (26). After washing and 10 min of incubation in saponin medium, cells were incubated for 30 min at room temperature with the appropriate concentration of FITC- or PE-conjugated cytokine-specific mAb. The following mAbs were used: PE-11B11 anti-IL-4 (PharMingen), PE-TRFK5 anti-IL-5 (PharMingen), FITC-JES5–16E3 anti-IL-10 (PharMingen), and FITC-labeled XMG1.2 (2). Cells were then washed with PBS/FCS in the absence of saponin to allow membrane closure. Data were collected on 20,000 CD4+ cells on an XL Coulter cytometer (Coultronics) and analyzed using CellQuest software (Becton Dickinson).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Evaluation of IgE production and endogenous IL-4 synthesis after neonatal induction of lymphoid chimerism in ß2m-deficient BALB/c mice

Neonatal injection of 108 semiallogeneic CB6 F1 SCs into BALB/c mice results in a host-vs-graft reaction that is characterized in vivo by an increased serum IgE level (27) which is dependent upon IL-4 synthesis (28). To assess the contribution of NK1.1+ T cells in priming for IL-4 production and IgE synthesis in this model, BALB/c ß2m+/- or ß2m-/- littermates were injected i.p. within the first 24 h of life with CB6 F1 SC. Mice were bled 4 wk later, and the serum IgE concentration was determined by ELISA. As shown in Figure 1GoA, no significant differences in serum IgE level can be demonstrated between both groups, although variations do occur among individual mice. The incidence of mice with a high serum IgE level in neonatal injected animals is equivalent between ß2m+/- and ß2m-/- BALB/c mice, at 73% and 82%, respectively. Next, we examined the kinetics of IgE production in serum. The results in Figure 1GoB show that the serum IgE concentration decreases rapidly from wk 4 to wk 6 and is maintained from wk 6 to wk 8. IgE levels in neonatal injected mice remain higher than those seen in normal mice, in which values were below the detection limit (0.1 µg/ml). Again, no significant difference is observed between the two cohorts of mice.



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  		FIGURE 1. Effect of ß2m expression in neonatal primed BALB/c mice on IgE serum levels. BALB/c heterozygous (ß2m+/-) or deficient (ß2m-/-) littermates were injected within the first 24 h of life with 108 CB6 F1 splenocytes. A, mice were bled at wk 4 (pooled data from four independent experiments). B, the kinetics of the serum IgE levels in ß2m+/- (n = 10) and ß2m-/- (n = 7) mice were examined. The serum IgE level was determined by ELISA, and the results are expressed as the mean IgE concentration (µg/ml) ± SD.

 
In addition to its role in IgE synthesis, IL-4 enhances the expression of MHC class II (29, 30) and low-affinity IgER (CD23) (31) on B cells. Therefore, we analyzed the intensity of the expression of these two molecules on splenic B cells in 4 wk old ß2m+/- or ß2m-/- littermate BALB/c mice that had been injected at birth with CB6 F1 SC. Simultaneously, the level of chimerism was tested by measuring the percentage of donor-derived cells using a H-2 Kb-specific mAb. As shown in Figure 2Go, IgE production in CB6 F1-injected ß2m-/- or ß2m+/- mice is associated with the presence of donor-derived cells in the spleen (5–10% of total SCs) as well as with a strong up-regulation of the cell surface expression of both MHC class II and CD23 on B cells. Again, there is no significant difference between the two groups.



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  		FIGURE 2. Flow cytometry analysis of chimerism and class II/CD23 expression on B cells in 4-wk-old mice. BALB/c heterozygous (ß2m+/-) or deficient (ß2m-/-) littermates were injected as described in Figure 1Go (6–9 mice per group) or left untreated (3 mice per group). Mice were sacrificed at 4 wk of age, and the serum IgE concentration was determined (A). Chimerism was analyzed on SCs using an H-2 Kb-specific mAb (B). The up-regulation of MHC class II I-E (C) and CD23 (D) expression was measured on B220+ cells, and the results are expressed as the mean fluorescence intensity. Bars represent the means and SDs of individual determinations. Only mice with a serum IgE level of >0.1 µg/ml are shown (70–90% of all injected neonates).

 
Taken together, our data show that a Th2-type immune response can be induced in the absence of host-derived ß2m-dependent T cells following a neonatal injection of normal semiallogeneic SCs. The level of chimerism is comparable in both ß2m-deficient and wild-type (wt) mice, and chimerism is necessary for the expression of the IL-4-dependent parameters.

Effect of ß2m expression in priming of Th2-type alloreactive CD4+ T cells

Adult BALB/c ß2m+/- or ß2m-/- littermates that had been injected at birth with CB6 F1 SCs or left untreated were all immunized s.c. with irradiated C57BL/6 SCs. Draining LNCs were collected 6 days later and restimulated with the indicated allogeneic or syngeneic class I-deficient APCs to selectively stimulate I-Ab-specific CD4+ T cells. The polarization of the T cell response was determined by measuring the cytokine production in 72-h culture supernatants. The data in Figure 3Go show that LNCs from primed BALB/c ß2m+/- or ß2m-/- littermates proliferate strongly in response to allogeneic but not syngeneic APCs. Interestingly, although the level of proliferation is comparable between uninjected control and CB6 F1-injected mice, the cytokine profile is clearly different. While alloreactive T cells from uninjected control mice produce a high amount of IFN-{gamma} but no IL-4, the T cells of CB6 F1-injected mice exhibit decreased IFN-{gamma} production that is associated with a strong expansion of IL-4-producing cells. In addition to IL-4, IL-10 production was also strongly up-regulated, which confirms the Th2 polarization of alloreactive CD4+ T cells in CB6 F1-injected mice (data not shown). Again, no difference could be observed between normal and ß2m-deficient BALB/c mice. Similar results were obtained using CB6 F1 or B6 SCs from ß2m+/+ mice as stimulators (data not shown).



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  		FIGURE 3. Proliferative and cytokine responses to the alloantigen of immune LNCs from neonatal injected or control mice. BALB/c heterozygous (ß2m+/-) or deficient (ß2m-/-) littermates were injected as described in Figure 1Go or left untreated. After 12 wk, mice from all groups were immunized s.c. with 60 x 106 splenocytes from C57BL/6 mice. LNCs from individual mice were collected 5 to 6 days later and cultured at 3 x 105 cells/well with irradiated SC APCs (3 x 105 cells/well) from ß2m-deficient C57BL/6 or BALB/c mice. For T cell proliferation, cells were cultured for 72 h and pulsed for the last 8 h of culture with [3H]TdR. Data are expressed as the mean of [3H]TdR incorporation, with background proliferation subtracted ({Delta} cpm). IFN-{gamma}, IL-4, and IL-10 production were assayed in 72-h culture supernatants by ELISA. The mean values of five mice per group are also shown (+). The results are from one representative experiment of three performed.

 
To further characterize the phenotype of individual I-Ab-reactive CD4+ T cells, immune LNCs that had been stimulated in vitro with B6 ß2m-/- APCs were analyzed at the single-cell level for the intracellular production of various cytokines after 7 days in culture. The results in Figure 4GoA show staining for IFN-{gamma} and IL-4 on CD4+ cells after restimulation with PMA/ionomycin. As expected from the quantitation of cytokine secretion, the I-Ab-specific CD4+ T cell response is polarized toward the Th1 phenotype (30–48% IFN-{gamma}+ cells) in control uninjected ß2m+/- or ß2m-/- BALB/c mice. Conversely, the induction of lymphoid chimerism in both wt and ß2m-deficient BALB/c mice results in strong skewing toward Th2 (13–30% IL-4+ cells). Among the IL-4-producing cells, 2 to 5% also produce IFN-{gamma} and therefore belong to the Th0 subset. Such cells are virtually absent in control BALB/c mice. The expansion of Th2-type cells is associated with a strong inhibition of the frequency of IFN-{gamma} single-positive cells that decreases from between 30 and 48% to between 3 and 9%. We also tested the expression of the two other Th2-associated cytokines, IL-5 and IL-10 (Fig. 4GoB). Although, the up-regulation of IL-5 synthesis is measured in culture supernatants (data not shown), few IL-5-producing cells are detected in CB6 F1-injected mice. Conversely, the frequency of IL-10-producing cells (8–21%) is markedly increased (10-fold) in chimeric mice as compared with uninjected control BALB/c mice and is not significantly different between ß2m-/- and wt controls.



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  		FIGURE 4. Single-cell analysis of the intracellular synthesis of cytokines. At 72 h, cells from the primary culture that had been restimulated with allogeneic APCs as described in Figure 4Go were expanded in complete medium; T cell blasts were collected after Ficoll separation at day 7. Before intracellular staining of the indicated cytokines, cells were stimulated with PMA and ionomycin for 4 h in the presence of brefeldin A for the last 2 h. Staining was performed as indicated in Materials and Methods, and acquisition was completed on CD4+ T cells. The results are expressed as the mean ± SD of five mice per group and are from one representative experiment of two performed.

 
In conclusion, these data show that a recall Th2- and Th0-type response can be induced in adult BALB/c mice that have been neonatally injected with semiallogeneic cells in the absence of ß2m-dependent T cells in the host. Interestingly, this expansion of Th2 cells is associated with a strong inhibition in Th1 cell development in CD4+ alloreactive T cells.

IgE production and Th2 cell priming in the complete absence of ß2m-dependent T cells

Although, it has been shown that the presence of T cells in the inoculum is not required for the induction of neonatal tolerance and its associated autoimmune syndrome (23), we cannot exclude the possibility that NK T cells present in the injected CB6 F1 splenocytes may contribute to the Th2-response in chimeric animals. To address this point, ß2m-/- BALB/c neonates were injected with CB6 F1 SCs that were or were not deficient for the ß2m gene. The results in Figure 5GoA demonstrate that a strong production of IgE in serum of 4-wk-old mice is observed in the complete absence of ß2m-dependent T cells. IgE synthesis is even higher in mice that have been injected with ß2m-deficient CB6 F1 SCs as compared with CB6 F1-injected animals. This increased production of IgE is associated with an augmentation of the splenomegaly in mice injected with ß2m-/- F1 SCs (Fig. 5GoB). Conversely, no difference in the up-regulation of MHC class II and CD23 molecules on B cells is seen between the two groups (Fig. 5Go, C and D).



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  		FIGURE 5. Th2 responses in the complete absence of ß2m-dependent cells. BALB/c ß2m-/- neonates were injected with normal or ß2m-deficient CB6 F1 SCs or left untreated. The serum IgE concentration (A), number of splenocytes (B), and up-regulation of class II I-E (C) and CD23 (D) expression on B cells were determined in 4-wk-old mice as described in Figure 2Go. The results are expressed as the mean ± SD of determinations from five to six mice per group. Results are from one representative experiment of three performed.

 
These data demonstrate that IgE synthesis and the up-regulation of I-E and CD23 on B cells, which are both dependent upon IL-4 synthesis in vivo, occur in the complete absence of ß2m-dependent T cells following a neonatal induction of lymphoid chimerism in mice.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Since the role of NK1.1+ T cells in the Th2 response and in the IL-4-dependent IgE production response has only been shown through use of a polyclonal stimulus such as anti-IgD (12), we have analyzed in the present paper the requirement for ß2m-dependent T cells in another model of polyclonal T-B cell interaction. A neonatal injection of semiallogeneic cells induces a state of chimerism that results in the absence of cytolytic alloreactivity (32) and promotes the differentiation of donor-specific CD4+ Th2 cells (28, 33, 34, 35). This allogeneic T-B cell interaction results in IL-4-dependent polyclonal activation of host Th2 and donor B cells, inducing the preferential production of IgG1 and IgE Abs (21, 22, 28, 36). Our data in the present paper demonstrate that even though these two models exhibit some similarities, they clearly differ in their requirement for ß2m-dependent T cells. First, IgE synthesis and the IL-4-dependent up-regulation of MHC class II and CD23 molecules on B cells are independent of ß2m expression in the host. Second, by measuring Th2-priming in adult mice, no difference in the induction of CD4 alloreactive Th2 cells could be observed between ß2m-/- mice and their wt control littermates. Finally, the Th2 response and IgE production are induced in the complete absence of ß2m-dependent T cells both in the host and in the inoculum. Therefore, using a variety of assays, we could not demonstrate diminished responses in mice with a disrupted ß2m gene in this model of Th2-mediated allogeneic interaction.

Although it has been shown that CD1-restricted T cells remain in MHC class II, ß2m-/- mice (37), the recent demonstration that IgE production to anti-IgD challenge is independent of CD1-dependent cells (38) indicates that the persistence of a residual population of CD1-restricted NK T cells in ß2m-/- mice is probably not involved in the generation of Th2 responses. Indeed, it has been shown that IL-4-producing cells are CD1-independent upon in vitro and in vivo stimulation with anti-CD3 in CD1-deficient mice. In contrast, the IgE response to anti-IgD was not modified in the same animals (38). Thus, the exact nature of the ß2m-dependent CD1-independent cells that are required for the IgE response to anti-IgD still remains to be characterized, but this cell population appears to be dispensable not only for the initiation of Th2 responses to protein Ags (13, 14, 15) and pathogens (14), but also in alloreactive Th2 responses. Although, mast cells (39), basophils (40), and eosinophils (41) can produce IL-4, several reports indicate that CD4+ T cells alone are sufficient for the generation of Th2 responses in vivo (42, 43).

In the model of induction of neonatal chimerism, it has been previously shown that host CD4+ Th2 cells progressively lose their reactivity toward the F1 semiallogeneic B cells, and that this unresponsiveness correlates with the disappearance of serum autoantibodies and with autoimmune pathology (35). In the present paper, we demonstrate that alloreactive Th2 cells persist in 10- to 12-wk-old chimeric animals, as shown by their capacity to mount a polarized Th2-type response upon subsequent strong immunogenic challenge with allogeneic cells. These results are in agreement with previous data showing that T cells that were able to react with donor B cells were still present in neonatally injected adult mice (44). More recently, it has been shown that alloreactive Th2 cells with a memory phenotype persisted for up to 12 wk after the neonatal induction of lymphoid chimerism (45, 46). By studying the phenotype of the alloreactive CD4+ T cells at the single-cell level, we show that Th2 (IL-4+/IFN-{gamma}-) and Th0 (IL-4+/IFN-{gamma}+) cells expand in neonatal primed animals, while these cells are almost absent in control uninjected mice. The increased frequency in Th2 cells is associated with a similar up-regulation in IL-10-producing CD4+ T cells, suggesting that both cytokines are coexpressed in the same cells. Conversely, the frequency of CD4+ IFN-{gamma}-producing Th1 cells is strongly reduced as compared with control mice. Thus, neonatal alloantigen exposure results in the selective expansion of CD4+ Th2 cells, which is associated with a long-lasting block of the Th1 response. Several lines of evidence indicate that the development of alloreactive IL-4- and IL-10-producing CD4+ T cells is probably responsible for the induction and maintenance of Th1 cell unresponsiveness in adult mice. First, it has been shown that both cytokines can synergize to inhibit cell-mediated immunity in vivo (47). Second, blocking endogenous IL-4 at the time of Th2 priming prevents the induction of neonatal tolerance to alloantigens (28, 36). Interestingly, the recovery of CTL activity was only evident when high doses of anti-IL-4 mAb were administered (28), indicating that a complete blockade of Th2 development was necessary to completely restore the Th1-dependent cytotoxic responses. Therefore, as suggested by others (45, 46, 48, 49), the induction of donor-specific CTL unresponsiveness is likely to involve Th2-dependent active regulatory mechanisms rather than the deletion or anergy of allogeneic CTL precursors. Indeed, it has been shown in this model that skin-graft rejection or acceptance was associated with enhanced Th1- or Th2-type responses, respectively (45).

The allogeneic interaction in this model involves both MHC class I and class II molecules. Although it is well accepted that MHC class I-reactive CTLs are undetectable in neonatally injected mice (32), the recent demonstration that CD8+ T cells can differentiate into noncytotoxic, IL-4-, IL-5-, and IL-10-producing cells (50) raises the question of whether such type 2 CD8+ T cells could play a role in the polyclonal activation of donor F1 B cells. Although some CD8+ T cells persist in ß2m-deficient mice (51), the demonstration that IgE production and Th2 cell priming occur normally in CB6 F1-injected ß2m-/- BALB/c mice strongly suggests that conventional, MHC class I-restricted, CD8+ T cells are dispensable for the Th2 response in this model. This possibility is further supported by the demonstration that IL-4-dependent IgE production can still be induced when donor F1 B cells do not express MHC class I molecules. Interestingly, the IgE response was even exacerbated and was associated with an increased splenomegaly in this latter situation. Since, recent reports indicate that CD8 T cells can regulate Th2 responses and IgE production in vivo (52, 53, 54, 55, 56), it is tempting to speculate that CD8+ T cells might be an important factor in controlling the alloreactive Th2 response in this model. Current experiments are in progress to address this issue.

In conclusion, our data demonstrate that Th2 cell development and IgE production are independent of ß2m expression in a model of polyclonal T-B cell interaction. Therefore, the early source of IL-4 that is required for the initiation of the alloreactive Th2 response does not involve ß2m-dependent NK1.1+ T cells. Thus, as has been shown by us and others in response to protein Ags (13, 15) and infectious microorganisms (14), ß2m-dependent T cells appear to be dispensable in the generation of alloreactive Th2 responses.


   Acknowledgments
 
We thank Marilyne Calise and Sylvie Pilipenko for their skillful technical assistance.


   Footnotes
 
1 This work was supported in part by grants from Etablissement Français des Greffes and Conseil Régional Région Midi-Pyrénées. G.F. is on a leave of absence from Ecole Nationale Vétérinaire de Toulouse, 31 076 Toulouse Cedex, France. Back

2 Address correspondence and reprint requests to Dr. Jean-Charles Guéry, INSERM U28, Hôpital Purpan, Place du Dr Baylac, 31 059 Toulouse Cedex, France. Back

3 Abbreviations used in this paper: ß2m, ß2-microglobulin; SC, spleen cell; CB6 F1, (BALB/c x C57BL/6)F1; PE, phycoerythrin; LNC, lymph node cell; wt, wild type. Back

Received for publication February 17, 1998. Accepted for publication April 16, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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